DOI: 10.1007/s11099-014-0047-4 PHOTOSYNTHETICA 52 (3): 404-412, 2014

Water stress and abscisic acid treatments induce the CAM pathway in the epiphytic fern Vittaria lineata (L.) Smith

B.D. MINARDI+, A.P.L. VOYTENA, M. SANTOS, and Á.M. RANDI

Plant Physiology Laboratory, Department of Botany, Federal University of Santa Catarina, Florianópolis, SC– 88049-900, CP 476, Brazil.

Abstract

Among various epiphytic ferns found in the Brazilian Atlantic Forest, we studied Vittaria lineata (L.) Smith (Polypodiopsida, Pteridaceae). Anatomical characterization of the leaf was carried out by light microscopy, fluorescence microscopy, and scanning electron microscopy. V. lineata possesses succulent leaves with two longitudinal furrows on the abaxial surface. We observed abundant stomata inside the furrows, glandular trichomes, paraphises, and sporangia. We examined malate concentrations in leaves, relative water content (RWC), photosynthetic pigments, and chlorophyll (Chl) a fluorescence in control, water-deficient, and abscisic acid (ABA)-treated plants. Plants subjected to drought stress (DS) and treated by exogenous ABA showed significant increase in the malate concentration, demonstrating nocturnal acidification. These findings suggest that V. lineata could change its mode of carbon fixation from C3 to the CAM pathway in response to drought. No significant changes in RWC were observed among treatments. Moreover, although plants subjected to stress treatments showed a significant decline in the contents of Chl a and b, the concentrations of carotenoids were stable. Photosynthetic parameters obtained from rapid light curves showed a significant decrease after DS and ABA treatments.

Additional key words: chlorophyll fluorescence; malate; morphoanatomy; photosynthetic pathway; pigments.

Introduction

Ferns are extensively distributed worldwide. These plants both land and water. They are climbers, thrive amid rocks, have developed remarkable adaptations in extreme envi- and they can be also epiphytes or hemiepiphytes, ranging ronments, being found in widely different habitats from tiny plants of a few millimeters to long, arborescent (Rathinasabapathi 2006). Many species are cosmopolitan forms reaching 20 m (Tryon and Tryon 1982). The and preferentially live in the tropical regions of the world, epiphytes comprise approximately 10% of the vascular especially in humid and shady forests (Rathinasabapathi flora (Kress 1986). They are found almost exclusively in 2006). Prado (2003) found approximately 13,000 species tropical forests, where they make up to 25% of species of ferns and lycophytes worldwide, out of which more (Nieder et al. 2001). According to Madison (1977), than 1,200 belong to Lycophyta and about 11,500 to epiphytic plants do not have direct connections with the Monilophyta (Pryer et al. 2004). ground. They only use phorophytes as a support, not as a Ferns are a very important group of plants which show source of nutrients. In an ecological context, epiphytism is well diversified ecological aspects. They have adapted to an interaction between plants in which the epiphytic

——— Received 3 June 2013, accepted 17 December 2013. +Corresponding author; fax: 0055-48-37218535; e-mail: [email protected]. Abbreviations: A – scaling constant for the height of the light curve; ABA – abscisic acid; AL – actinic light; Car – carotenoids; DM – dry mass; DS – drought stress; ETR – electron transport rate; ETRmax – maximum electron transport rate; FM – fresh mass; F0 – minimum fluorescence; Fm – maximum fluorescence; Fv – variable fluorescence; Fm'– minimum fluorescence of a light-adapted leaf; F' – fluorescence yield of a light-adapted leaf; FLM – fluorescence microscopy; Fv/Fm – maximum photochemical efficiency of PSII; I – irradiance; Iopt – optimal irradiance for the saturation of photosynthesis; kw – scaling constant for the x-axis of the light curve; LM – light microscopy; OAA – oxaloacetic acid; PAM – pulse-amplitude modulation; P – photosynthesis; PEG – polyethylene glycol; PEPC – phosphoenolpyruvate carboxylase; RLC – rapid light curve; RWC – relative water content; SEM – scanning electron microscopy; SL – saturating light pulses; TM – turgid mass; ФPSII – actual photochemical efficiency of PSII. Acknowledgments: The first authors would like to acknowledge CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the scholarship (Rede em Epífitas de Mata Atlântica: Sistemática, Ecologia E Conservação, CAPES, PNADB, 2009). Á.M. Randi and M. Santos would like to acknowledge CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the research grants.

404 CAM PATHWAY IN VITTARIA LINEATA plant is dependent only on the substrate supplied by the 1983, Sinclair 1984, Ravensberg and Hennipmam 1986, host plant (phorophyte); otherwise, they obtain nutrients Rut et al. 2008). Increase in titratable acidity was found in directly from atmospheric moisture, without emitting Dictymia J. Sm (Griffiths 1989) and V. lineata (Carter and haustorium structures (Bennett 1986). Vittaria lineata (L.) Martin 1994). Recently, Martin et al. (2005) showed some Smith (Pteridaceae) is an epiphytic fern found in the evidence of the CAM pathway in Vittaria flexuosa Fée and Brazilian Atlantic Forest. It is an exclusively epiphytic Anetium citrifolium (L.) Splitg., but not in V. lineata. On species without petioles and presenting laminar leaf the other hand, Martin et al. (2005) suggest the CAM (10–50 cm), simple, pendulous; linear sori is in a row pathway for V. flexuosa and A. citrifolium, which may be between the rachis and the margin and monolete spores also a sporadic phenomenon in V. lineata, thus explaining (Tryon and Tryon 1982, Smith et al. 2006). Known the disparity between the findings of Martin et al. (2005) popularly as "shoe cord fern", this species grows and those of Carter and Martin (1994). However, the preferentially in damp woods as epiphytes on tree trunks titratable acidity may not be enough to establish the or in the canopy (Tryon and Tryon 1982). Based on its presence of the CAM pathway because substances other beauty and hardiness, it is an ornamental plant widely used than malate may influence the pH of the samples. Zotz and in gardens. Ziegler (1997) studied the occurrence of CAM in different The epiphytism favors the capture of light radiation, vascular epiphytes in a central Panamanian forest via 13C but limits the availability of water. The CAM pathway is isotope and they verified C3 metabolism in V. lineata. one of the most important adaptations of some epiphytic CAM was initially verified in the epiphytic fern ferns to water deficit. It enables CO2 assimilation at Vittaria lineata (L.) Smith by Carter and Martin (1994), increased water deficiency and high irradiance stress but the presence of this pathway in V. lineata has never (Benzing 1986, 1990; Kluge et al. 1989). In plants that been confirmed (Zotz and Ziegler 1997, Martin et al. show the CAM pathway, CO2 enters the mesophyll leaf 2005). We hypothesized that V. lineata could change its tissue through open stomata predominantly at night and is mode of carbon fixation from C3 to the CAM pathway in fixed in the reaction catalyzed by phosphoenolpyruvate response to water deficit and exogenous application of carboxylase (PEPC) in the cytosol. The oxaloacetate ABA. Therefore, this study aimed to analyze the effects of (OAA) resulting from PEPC activity is reduced to malic water deficit and ABA application on relative water acid, which is accumulated in the vacuole at nighttime content (RWC), photosynthetic pigments, rapid light (Lüttge 1993, Nimmo 2000). curves, and fluctuations in the malate contents in leaves in In the Polypodiaceae, the CAM pathway was found in order to evaluate the physiological adaptations of five species of Pyrrosia and in Platycerium bifurcatum V. lineata to stress. (Hew and Wong 1974, Wong and Hew 1976, Winter et al.

Materials and methods

Plant collection: Plants of Vittaria lineata (L.) Smith were they were irrigated with Hoagland's solution (20%, v/v) collected from the natural habitat in the Environmental (Hoagland and Arnon 1938). After the acclimation period Conservation Unit “Desterro” (UCAD), located in the of at least six months, the plants were divided into three northwestern part of the island of Florianópolis, Santa groups, each subjected to a different conditions: (1) control Catarina, Brazil (27°31'50''S, 48°30'44''W, an area of 495 plants irrigated daily for two weeks, (2) plants subjected to ha, which is about 1% of the area of the island). The unit DS for a period of one week (without irrigation), and (3) is managed by the Federal University of Santa Catarina plants irrigated 5 times a week with a solution of 10 µM (UFSC), Florianópolis, Santa Catarina, SC, Brazil. ABA for a period of 15 d.

Plant acclimation: Plants were transported to the green- Structural analysis of leaves: From the leaves in vivo, we house of the Botany Department of the Federal University cut freehand cross sections, both longitudinal and para- of Santa Catarina, where they grew for a minimum of six dermic, with a razor blade, using polystyrene as support months. The plants were placed in brackets on the walls. material. These sections were placed on a glass slide with During the testing period, the daily temperature of the water and covered with a cover slip. Histochemical stain- greenhouse varied between 20–30°C, and at night, it was ing was carried out using Lugol's solution for identification kept around 15°C. The relative humidity was monitored of starch (Jensen 1962), FeCl3 for phenolic compounds and ranged between 60–80%. The PPFD inside the green- (Johansen 1940), Sudan IV for lipophilic substances house alternated between 43 and 85 µmol(photon) m–2 s–1. (Gerlach 1984), and acidified phloroglucinol for lignin Irradiance was measured at the mid-region of the plants (Costa 1982). The stomata count was performed in with an LI-190 sensor (LI-COR Instruments, USA) con- triplicate in the median region of the abaxial surface. The nected to the LI-250 light meter (LI-COR Instruments, stomata count was done with a 20× objective of a Leica USA) placed close to plants. The plants were watered daily DM2500 optical microscope (LM) on images captured with distilled water to retain high humidity. Once a week, with a digital camera (Leica DFC295, Leica, Germany).

405 B.D. MINARDI et al.

Structural analysis of the leaves was also performed on 98ºC, kept in water bath at 98ºC for 10 min, and samples fixed in 2.5% glutaraldehyde in 0.1 M sodium subsequently centrifuged at 3,500 × g for 10 min. The phosphate buffer (pH 7.2), washed in the same buffer, precipitate was discarded and the extracts kept at room dehydrated in ethanol series, and preserved in ethanol 70% temperature. Aliquots of 100 µL were used to quantify the (v/v) (Ruzin 1999). Some samples were infiltrated in a malate through the K-LMALL enzymatic kit (Megazyme mixture of polyethylene glycol (PEG) 1500 and 70% International Ireland Limited, County Wicklow, Ireland). ethanol (1:1) for 24 h in an oven at 60°C. They were again The detection of malic acid requires two enzymatic incubated in an oven for 24 h in pure PEG 1500. Following reactions. The first reaction is catalyzed by L-malate this incubation, the samples were embedded in PEG 1500. dehydrogenase, whereby the malic acid is oxidized to Other samples were infiltrated in methacrylate (Jung's OAA by NAD+. However, since the reaction balance is Historesin, Leica, Wetzlar, Germany). The samples favorable to malic acid and NAD+, a further reaction is preserved in ethanol 70% (v/v) were dehydrated in ethanol needed to keep the NADH product, and this is achieved by series up to 96% (v/v) and processed according to the conversion of OAA to L-aspartate and α-ketoglutarate manufacturer’s instructions. The samples were embedded (2-oxoglutarate) in the presence of a large excess of in PEG and methacrylate, and afterwards, they were L-glutamate by glutamate oxaloacetate transaminase. The sectioned by rotary microtome (Leica RM 2125 RT; Leica, amount of NADH formed in the reaction described above Wetzlar, Germany) and stained with Astra blue (Bukatsh corresponds stoichiometrically to the amount of malic 1972) or toluidine blue (O'Brien et al. 1965) and safranin acid. Thus, NADH may be measured by the increase in (Kraus and Arduin 1997). absorbance. The analyses were performed spectrophoto- The samples were examined under LM (Leica metrically (SP-220, Biospectro, Brazil) at 340 nm, in DM2500; Leica, Wetzlar, Germany), and images were triplicate, and the concentration of malate was expressed captured with an attached digital camera (Leica DFC295, in µmol g–1(dry mass, DM). Leica, Wetzlar, Germany). For structural analysis of the leaves in vivo by fluo- Determination of relative water content (RWC): After rescence microscopy (FLM), we used an Olympus BX41 each treatment, leaf discs (for each replicate n = 3) of 1.0 g fluorescence microscope (Olympus, Tokyo, Japan) with a of fresh mass (FM) were cut and placed in flasks mercury-vapor lamp as the light source (HBO 100) and containing distilled water for 180 min under the irradiance blue color filter U-MWU2 (excitation: 330–385 nm, of 20 µmol(photon) m–2 s–1, at 25 ± 2°C to obtain the turgid emission: 420 nm), red color filter U-MWG2 (excitation: mass (TM). Then, the leaf segments were dried in an oven 510–550 nm, emission: 590 nm), green color filter at 60°C for 24 h and subsequently weighed to obtain DM. U-MWB2 (excitation: 460–490 nm, emission: 520 nm), The RWC values of fully expanded, mature leaves were and triple filter (excitation: 330–385 nm, 460–490 nm, expressed by the equation: 510–550 nm; emission: 420 nm, 520 nm, 590 nm). The RWC = [(FM – DM)/(TM – DM)] × 100 (Barrs 1962). images were captured with a QColor 3C digital camera (Q-imaging, Surrey, BC, Canada). Photosynthetic parameters by Chl a fluorescence: Chl fluorescence was evaluated using a pulse amplitude- Ultrastructural analysis of leaves: The prepared leaf modulated fluorometer (Diving-PAM, Walz, Effeltrich, samples were subjected to total dehydration in ethanol Germany), equipped with an optic fiber 5.5 mm in series and kept in ethyl ether for 48 h at 20°C. Afterwards, diameter and a blue diode (470 nm) as the light source. the sample containers were opened and kept in laminar During the testing period, the recorded temperature was flow to favor complete evaporation of the ether. The 25 ± 2°C. The analyses were carried out between 10:00 samples were then placed on aluminum brackets with the and 12:00 h. All plants were acclimated in the dark for 25 aid of double-sided carbon tape. Leaf samples were min before the application of the first pulse of saturating metalized with 30 nm of gold in a Bal-Tec CED 030 light. The experiments were performed in triplicate. (n = 3). metalizer (Bal-Tec AG, Balzers, Liechtenstein). Scanning Ten rapid curves were obtained for each replicate. The electron microscope (SEM) JEOL JSM - 6390LV (JEOL, minimum fluorescence (Fo) was obtained by submitting Tokyo, Japan) was used to analyze the samples. samples to a measured modulated light [0.1 μmol(photons) – –1 m ²s ], and the maximum fluorescence (Fm) in the dark- Analysis of malate content: Malate contents were acclimated samples was obtained just after applying a SL evaluated via enzymatic analyses according to Möllering [9,000 µmol m–2 s–1]. Using the option “Rapid Light (1985) in order to verify the difference in malate Curve” (RLC) in the Diving-PAM software, the whole concentration (Δ malate) between night and day. Three light curves were obtained by applying a series of eight samples (1.0 g each) were taken from fresh leaf at 6:00 and 0.3s saturating light pulses (SL, 9,000 µmol(photons) 18:00 h from each treatment. They were immediately m–²s–1), each one, followed by exposure to crescent actinic immersed in liquid nitrogen (–192°C) to stop all enzymatic light (AL) [from 2 to 2250 µmol(photons) m–2 s–1]. The reactions until analysis was carried out. For the extraction, parameters, i.e., electron transport rate (ETR), were samples were homogenized in 10 ml of distilled water at calculated using the WinControl software (V.3.18) (Walz,

406 CAM PATHWAY IN VITTARIA LINEATA

Germany) according to Genty et al. (1989). During the Statistical analysis: Data were analyzed by Excel and RLC, each SL and AL period produced a light-adapted BioEstat software. Data were expressed as mean ± standard maximum fluorescence (Fm') and a light-adapted minimum deviation. The analysis of variance (multifactor ANOVA) fluorescence (F'). The ETR between PSII and PSI was was followed by the mean comparison test (Tukey 5%) for estimated using the following equation: ETR = ФPSII × data with normal distribution and homoscedasticity (Zar PPFD × 0.5 × 0.84, where ФPSII is the effective photo- 1996). To quantitatively compare RLCs using parametric chemical quenching yield of PSII obtained by statistics, some descriptive parameters were used: maxi- (Fm' – F')/Fm', PPFD is the photosynthetic photon flux mum electron transport rate (ETRmax) and optimal irradi- density during exposure to the actinic light, 0.5 is related ance for the saturation of photosynthesis (Iopt). ETR data to the incident photon between PSII and PSI, and 0.84 is were plotted as rapid light curves (RLC = ETR vs. I, where the fraction of incident quanta absorbed by the leaf I are the irradiation pulses applied to draft the curves). (Bjorkman and Demmig 1987, Schreiber et al. 2004, Both parameters were plotted in a waiting-in-line equation White and Critchley 1999, Runcie and Durako 2004, (y = x e–x) using an Excel macro (Microsoft Office Excel Ralph et al. 2005). 2010) (adapted from Ritchie 2008).The empirical model for ETR vs. I was first used by Gloag et al. (2007), who Extraction and quantification of photosynthetic pig- applied a suitable mathematical model for photosynthesis: –k I ments: After determination of the RLC, photosynthetic ETR= A kw I e w (adapted from Gloag et al. 2007 and pigments were analyzed according to Lichtenthaler (1987) Ritchie 2008).The equivalent form for this first equation using samples of 1.0 g of leaf FM (n = 3). For extraction, –k I is: ETR = ETRmax kw I e w . The ETRmax (A) occurs at an leaf samples were taken from liquid nitrogen and immedi- irradiance value of 1/k , the optimal irradiance for the ately homogenized in 10 ml of aqueous acetone (80%, v/v) w saturation of photosynthesis (I ). The adjusted values for for 5 min, using a mortar. The homogenate was centrifuged opt constants A (scaling constant for the height of the curve) for 10 min at 3,500 × g, and the volume was adjusted to and k (scaling constant for the axis X) were determined 10 ml. The absorbances at 663, 646, and 470 nm were w using the equation described by Gloag et al. (2007). analyzed with a UV-VIS spectrophotometer (SP-220, Biospectro, Brazil).

Results

Structure and ultrastructure of leaves: The leaf of treatments and application of ABA compared with the Vittaria lineata exhibited the average thickness of control plants, where the values were kept high (Table 1). 1.05 mm. In the leaf crosssections, we noted the presence In V. lineata, the control plants showed insignificant of two linear furrows on the abaxial surface (Fig. 1A). differences in malate contents between 6:00 and 18:00 h Inward, the mesophyll consisted of palisade and spon- (Fig. 2), presenting a low value of Δ malate [0.65 µmol geous parenchyma. Three meristeles were observed in the g–1(DM)]. This result proved no evidence of a CAM leaf, one central and two lateral corresponding to the pathway in the control plants. On the other hand, when the regions, where the furrows were linearly located (Fig. 1A). plants were subjected to DS for seven days and exogenous Stomata were observed only in the furrows on the abaxial application of ABA for a period of 15 d, an increase in the surface, showing the characteristic hypostomatic leaf, and concentrations of Δ malate was evidenced (Fig. 2). Daily occurred at the average concentration of 113 stomata per significant differences occurred between the night and day mm2. (Fig. 1B). In the epidermis of the linear furrow, a malate concentration in both treatments, showing a noc- large number of uniseriate and unbranched paraphyses turnal acidification and revealing an upregulation of the were seen, in addition to sporangia sori in a single row CAM pathway in response to different types of stress. In parallel to each leaf edge (Fig. 1B). Frontal view of the the leaves of plants subjected to DS and ABA, the daily epidermal surface in SEM revealed that the cuticle was difference in the concentration of night and day malate smooth, but with wax deposits (Fig. 1C). The FLM image (Δ malate) was 140 and 187 times greater, respectively. (green excitation: 460–490 nm, emission: 520 nm) Therefore, plants treated with ABA showed the greatest confirmed the presence of stomata having a red color as a accumulation of malate during nighttime, indicating a high result of chloroplasts in guard cells (Fig. 1D). V. lineata rate of nocturnal fixation of atmospheric CO2. leaves present polocytic type of stomata characterized by a subsidiary cell in a horseshoe shape. The anticlinal walls Photosynthetic pigments and RLC analysis: The of the epidermal cells are sinuous (Fig. 1E). In the contents of Chl a and total Chl were significantly lower epidermis of the furrow, glandular trichomes were under DS and ABA when compared with the control treat- observed to secrete substances of a lipid nature, as ment in leaves of V. lineata. Moreover, the content of Chl b evidenced by positive reaction to Sudan IV (Fig. 1F). and carotenoids (Car) after ABA treatment did not differ from the control. Car contents remained stable, even under RWC and malate content: Statistically insignificant diffe- stress treatments (Fig. 3). rence in RWC was observed independently of water-deficit

407 B.D. MINARDI et al.

Fig. 1. Cross sections of Vittaria lineata leaf in LM showing adaxial epidermis, abaxial epidermis, furrow, mesophyll consis- ting of parenchyma tending to palisade and spongy parenchyma, paraphyse, stomata, and three vascular tissues of the meristele type (A). SEM image of the adaxial epidermal surface of the leaf: furrow and sporangia inside the furrow indicated by arrows (B). SEM image of the abaxial epidermal surface of the leaf (C). Cross sections of the leaf in FLM (excitation: 460–490 nm, emission: 520 nm) showing stomata inside the furrow (red) and the cuticle (green) indicated by arrows (D). LM image of the epidermal abaxial surface of the leaf after reaction to safranin, showing polocytic stomata with subsidiary cells in a horseshoe shape and glandular trichome (E). Front view of the inner wall of the furrow showing glandular trichomes with positive reaction to Sudan IV, indicating the presence of lipids (F). ab – abaxial epidermis, ad – adaxial epidermis, ae – aerenchyma, ct – cuticle, epidermal cell, f – furrow, gc – guard cell p – paraphyse, pp – palisade parenchyma, sc – subsidiary cell, sp – spongy parenchyma, st – stomata, spr – sporangia, t – trichome, vt – vascular tissue of the meristele type.

Table 1. The changes of the relative water content (RWC) in leaves of Vittaria lineata under different treatments: control, drought stress (DS, 7 d), and 10 µM abscisic acid (ABA) (15 d). Lowercase letters indicate the groups differentiated by ANOVA followed by Tukey’s test, p> 0.05 (n = 3).

Treatment RWC [%]

Control 92.36 ± 0.25a DS 91.69 ± 0.48a ABA 91.45 ± 0.44a

In V. lineata, DS and ABA caused a reduction of 19.4 were also observed (Table 2). Rapid light curves in Fig. 4 and 29.1% in Iopt, respectively (Table 2). A decline in the show decrease in ETR of plants under DS and ABA ETRmax under DS treatments (26.2%) and ABA (19.4%) treatment.

Discussion

The succulent leaf has been described as a basic feature of with large vacuoles and an aerenchyma. Sensu lato, plants that assimilate carbon fixation through CAM aerenchyma is simply parenchymatous tissue with a large metabolism (Neales and Hew 1975). Vittaria lineata volume of intercellular space (Drew et al. 2000). The presents succulent leaves with two longitudinal furrows on presence of aerenchyma in leaves of xerophytic species of the abaxial surface, where abundant stomata are located in the Selaginella was verified almost a century ago by Uphof a protected area, which possibly represents a microclimate (1920). In this paper, we propose that the aerenchyma in that is intermediate to the internal and external environ- V. lineata could hold water vapor and accumulate CO2 ment of the leaf blade. Such a stomata location allows the from nocturnal respiration for photosynthetic activity creation of a humid microclimate within the longitudinal during the day. In V. lineata leaves, stomata are located furrows to maintain water during recurring periods of only in the furrows, which also have paraphyses, trichomes drought. The epidermal cells show sinuous anticlinal and sporangia. The presence of other structures in the walls, with thick cuticle. Hietz and Briones (1998) epidermis of the furrows could hinder the stomata CO2 reported that these features are commonly found in uptake. Actually, the mechanisms for CO2 accumulation in epiphytic ferns and are among attributes responsible for aerenchyma occur in plants living in habitats with stressful maintaining water during recurring periods of drought. environments. In the aquatic plant Lobelia dortmanna L. Vittaria lineata leaves show a parenchyma tending to the (Campanulaceae), Pedersen and Sand-Jensen (1992) show palisade, a spongeous parenchyma characterized by cells that the concentration of CO2 in its lacunae increases

408 CAM PATHWAY IN VITTARIA LINEATA

Fig. 2. Daily fluctuation (6:00–18:00 h) in malate concentration and diurnal malate fluctuations (Δ malate) in leaves of Vittaria lineata under different treatments: control, drought stress (DS, 7 d), and ABA-treated leaves (15 d) (10 μM). Lowercase letters indicate the groups differentiated by ANOVA followed by Tukey’s test, p>0.05 (n = 3). ABA – abscisic acid; DM – dry mass; DS – drought stress.

Fig. 3. Chlorophyll (Chl) a, Chl b, total Chl, and carotenoid (Car) contents in Vittaria lineata leaves under different treatments. Mean ± SD (n = 3). Lower- case letters indicate the groups differentiated by ANOVA followed by Tukey’s test, p>0.05 (n = 3). ABA – abscisic acid; DM – dry mass; DS – drought stress.

Table 2. Rapid light curve (RLC) parameters plotted as electron transport rate (ETR) vs. irradiance (I) in sporophytes of Vittaria lineata under different treatments. Means followed by the same letter are not significantly different. Lowercase letters indicate the groups differentiated by ANOVA followed by Tukey’s test, p>0.05 (n = 3). A – scaling constant for the height of the light curve; ETRmax – maximum electron transport rate; Iopt – optimal irradiance; kw – scaling constant for the x-axis of the light curve.

Parameter Control Water deficit ABA

–2 –1 a b b Iopt [µmol(photon) m s ] 1.653 ± 59 1.332 ± 83 1.172 ± 23 –2 –1 a b b ETRmax [µmol(electron) m s ] 97 ± 4 72 ± 6 78 ± 4 kw 0.0006 ± 0.0001 0.0007 ± 0.0001 0,0008 ± 0.0001 A 259 ± 14 190 ± 22 220 ± 19 Correlation coefficient r 0.98 0.99 0.97 n samples/curves 3/30 3/30 3/30

23 times of ambient atmospheric concentration, ranging Early responses usually help the plant to survive, while it from about 0.3% during the day to about 0.7% at night. acclimates, by the accumulation of certain new meta- Similar results were obtained from Typha latifolia L. bolites. They endow the plant with the structural capa- (Typhaceae) (Constable et al. 1992), where the gas in the bilities required to improve its function under DS (Pinhero leaf aerenchyma ranged from ambient CO2 contents et al. 1997). In the present paper, we verified the increase around noon to about 0.6% of CO2 (18 times higher than in Δ malate, when plants of V. lineata were subjected to ambient) in the early morning. water depletion and exogenous application of ABA. The responses of plants to DS are highly complex. Therefore, both ABA and DS induced CAM pathway in

409 B.D. MINARDI et al.

Fig. 4. Rapid light curve (RLC) plotted as ETR vs. irradiance in sporophytes of Vittaria lineata under different treatments: Mean ± SD (n = 3). Lowercase letters indicate the groups differentiated by ANOVA followed by Tukey’s test, p>0.05 (n = 3). ABA – abscisic acid; DS – drought stress.

V. lineata. The accumulation of malate at nighttime is a (N'Soukpoé-Kossi et al 1999). According to Maxwell and characteristic of the CAM pathway (Chu et al. 1990, Johnson (2000), the decrease of RLC under stress indicates Cushman and Borland 2002, Rut et al. 2008, Freschi et al. photoinhibition of PSII. Drought stress is known to inhibit 2010). Thus, our data suggest that V. lineata seemed to photosynthetic activity in tissues as a consequence of an change its mode of carbon fixation from C3 to the CAM imbalance between light capture and its utilization; pathway in response to DS and exogenous application of downregulation of PSII activity results in an imbalance ABA. This response was observed earlier by Chu et al. between the generation and utilization of electrons (Peltzer (1990) who observed that the facultative halophyte et al. 2002). From the data obtained in our study, we Mesembryanthemum crystallinum shifts its mode of suggest that DS and application of exogenous ABA carbon assimilation from the C3 pathway to CAM in negatively regulated photosynthesis or the rate of electron response to DS and ABA. Plants exhibiting this behavior transport in leaves of V. lineata. No significant differences are referred to as facultative CAM in contrast to obligatory between treatments were observed in Car contents in CAM, where the metabolism is not dependent on leaves of V. lineata after DS and ABA treatment. Tausz et environmental or developmental factors (Chu et al. 1990). al. (2001), also observed that the amount of Car was not According to Horton (1971), exogenous ABA initiates altered by different treatments of water restriction in stomata closure in leaves of angiosperms. In contrast, relation to the control treatment in most ferns. Car are part Brodribb and McAdam (2011) and McAdam and Brodribb of ROS detoxification in plants under DS. However, the (2012) showed that lycophyte and fern stomata lack key degree, to which the activities and amounts of antioxidant responses to ABA and to the epidermal cell turgor. enzymes increase under DS, varies among plants, depen Brodribb and McAdam (2011) observed that the stomata ding on the species, the development, and the of the fern species, Pteridium esculentum, showed no metabolic state of the plant, as well as the duration and response to high xylem ABA concentration, despite the intensity of the stress (Reddy et al. 2004). accumulation of high contents of ABA in the leaves. In the present paper, we observed an interesting leaf In V. lineata, DS and exogenous application of ABA anatomy in V. lineata. It could maintain leaf water balance did not alter the RWC when compared with the control and probably allow the plant to accumulate nocturnal CO2 plants. Both treatments led to a decrease in the contents of in aerenchyma as a strategy to compensate for the Chls, as well as ETRmax, Iopt, and, consequently, the RLC restriction in stomata number and localization, which of V. lineata leaves. The decrease in Chl content in plants could limit CO2 assimilation. Our data demonstrated that under stress has been previously described in angiosperm V. lineata could change its mode of carbon fixation from species (Asharf et al. 1994, Jaing et al. 1994) and epiphytic C3 to the CAM pathway in response to DS and exogenous ferns in a Mexican rainforest, i.e., Polypodium plebeium application of ABA. We suggest that controversial data on Schltdl. & Cham., Elaphoglossum petiolatum (Sw.) photosynthetic pathway of V. lineata (Carter and Martin Urban, Phlebodium areolatum (Humb. and Bonpl. ex 1994, Zotz and Ziegler 1997, Zotz 2004, Martin et al. Willd.) J. Sm., and Asplenium cuspidatum Lam. (Tausz et 2005) could be attributed to the presence of the facultative al. 2001). In barley leaves, ABA inhibited the photo- CAM pathway induced by DS and ABA. synthetic process, as evidenced by lower values of Fv/Fm

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